Fluorescent nucleoside analogs and combinatorial fluorophore arrays comprising same

Abstract

The present invention provides fluorescent nucleoside analogs which comprise a fluorescent cyclic compound joined to a carbon of a sugar molecule such as pentose, hexose, ribose or deoxyribose or analogs thereof in either an alpha or beta configuration. The subject compounds are useful as probes in the study of the structure and dynamics of nucleic acids and their complexes with proteins. In addition, the subject compounds are useful in any technique which uses labeled oligonucleotides for detection. Non-fluorescent spacer molecules in which a cyclohexane, cyclohexene, decalin, or benzene is joined to a carbon of a sugar moiety such as pentose, hexose, ribose or deoxyribose are also provided. Also provided are the 5' dimethoxytrityl-3'-O-phosphoramidite derivatives, suitable for incorporation into oligonucleotides by automated synthesizers. Combinatorial fluorophore array (CFA) libraries comprising oligomers of the subject nucleoside analogs attached to one or more solid supports are also provided as are methods of selecting fluorophores from the CFA libraries. The present invention also provides oligonucleotide analogs comprising one or more of the subject nucleoside analogs in place of a DNA or RNA base.

What is claimed is:  
     
       1. A method of selecting a fluorophore suitable for use in labeling a nucleic acid molecule which comprises: constructing a combinatorial fluorophore array library (CFA) which comprises multiple solid supports or multiple locations on a solid support, wherein each support or location has attached thereto an oligomer comprising a nucleoside analog, said nucleoside analog being a fluorescent cyclic compound joined to a carbon at the C-1 position of a sugar moiety in either an α or β configuration wherein said sugar moiety is a pentose or hexose, and selecting a fluorophore emitting the most intense florescence or emitting a specific wavelength of light. 
     
     
       2. The method according to  claim 1  wherein said sugar moiety is a ribose or deoxyribose. 
     
     
       3. The method according to  claim 1  wherein the fluorescent cyclic compound is an oligomer of varying length selected from the group consisting of oligothiophene, oligobenzothiophene, oligo(phenylene vinylene), and oligo(phenylene acetylene). 
     
     
       4. The method according to  claim 3  wherein the fluorescent cyclic compound has an oligomer length of from about 1 to about 16. 
     
     
       5. The method according to  claim 3  wherein the oligothiophene is a terthiophene or a sexithiophene. 
     
     
       6. The method according to  claim 3  wherein the oligobenzothiophene is a benzoterthiophene or a terbenzothiophene. 
     
     
       7. The method according to  claim 3  wherein the oligo(phenylene vinylene) is dimethylamino stilbene or styrylstilbene. 
     
     
       8. The method according to  claim 3  wherein the oligo(phenylene acetylene) is diphenylacetylene or phenyl(ethynyl)diphenylacetylene. 
     
     
       9. The method according to  claim 1  wherein the fluorescent cyclic compound is selected from the group consisting of p-terphenyl, perylene, azobenzene, phenazine, napthalene, phenanthroline, acridine, thioxanthrene, chrysene, rubrene, coronene, cyanine, perylene imide, and perylene amide. 
     
     
       10. The method according to any one of claims  1 - 9  wherein the combinatorial fluorophore array library further comprises one or more unlabeled nucleosides wherein the one or more unlabeled nucleosides are positioned 5′ or 3′ to the fluorescent nucleoside analogs or interspaced between the fluorescent nucleoside analogs. 
     
     
       11. The method according to any one of claims  1 - 9  wherein the CFA library further comprises one or more non-fluorescent nucleoside analogs selected from the group consisting of cyclohexene-2-deoxyriboside, cyclohexane-2-deoxyriboside, decalin-2-deoxyriboside, and benzene-2-deoxyriboside wherein said one or more no fluorescent nucleoside analogs are interspaced between the fluorescent nucleoside analogs. 
     
     
       12. The method according to  claim 10  wherein the CFA library further comprises one or more non-fluorescent nucleoside analogs selected from the group consisting of cyclohexene-2-deoxyriboside, cyclohexane-2-deoxyriboside, decalin-2-deoxyriboside, and benzene-2-deoxyriboside wherein said one or more non-fluorescent nucleoside analogs is interspaced between the fluorescent nucleoside analogs or between the fluorescent nucleoside analogs and the and unlabeled nucleosides. 
     
     
       13. A method of identifying a fluorophore emitting large Stokes shifts which comprises (a) constructing a combinatorial fluorophore array library which comprises multiple solid supports or multiple locations on a solid support, wherein each support or location has attached thereto an oligomer comprising a nucleoside analog, said nucleoside analog being a fluorescent cyclic compound joined to a carbon at the C-1 position thereof of a sugar moiety in either an α or β configuration and wherein said sugar moiety is one of pentose or hexose, (b) exciting the library at short wavelengths, and (c) selecting a fluorophore which emits light at a much longer wavelength. 
     
     
       14. The method of  claim 13  wherein the sugar moiety is ribose or deoxyribose. 
     
     
       15. The method according to  claim 13  wherein the fluorescent cyclic compound is an oligomer of varying length selected from the group consisting of oligothiophene, oligobenzothiophene, oligo(phenylene vinylene), and oligo(phenylene acetylene). 
     
     
       16. The method according to  claim 15  wherein the fluorescent cyclic compound has an oligomer length of from about 1 to about 16. 
     
     
       17. The method according to  claim 15  wherein the oligothiophene is a terthiophene or a sexithiophene. 
     
     
       18. The method according to  claim 15  wherein the oligobenzothiophene is a benzoterthiophene or a terbenzothiophene. 
     
     
       19. The method according to  claim 15  wherein the oligo(phenylene vinylene) is dimethylamino stilbene or styrylstilbene. 
     
     
       20. The method according to  claim 15  the oligo(phenylene acetylene) is diphenylacetylene or phenyl(ethynyl)diphenylacetylene. 
     
     
       21. The method according to  claim 13  wherein the fluorescent cyclic compound is selected from the group consisting of p-terphenyl, perylene, azobenzene, phenazine, napthalene, phenanthroline, acridine, thioxanthrene, chrysene, rubrene, coronene, cyanine, perylene imide, and perylene amide. 
     
     
       22. The method according to any one of claims  13 - 21  wherein the combinatorial fluorophore array (CFA) library further comprises one or more unlabeled nucleosides wherein the one or more unlabeled nucleosides are positioned 5′ to 3′ to the fluorescent nucleoside analogs or interspaced between the fluorescent nucleoside analogs. 
     
     
       23. A method of identifying a fluorophore involved in energy transfer which comprises (a) constructing a combinatorial fluorophore array library (CFA) which comprises multiple solid supports or multiple locations on a solid support, wherein each support location has attached thereto an oligomer comprising a nucleoside analog, aid nucleoside analog being a fluorescent cyclic compound joined to a carbon at the C1 position of a sugar moiety in either an α or β configuration wherein said sugar moiety is a pentose or hexose, and one or more non-fluorescent nucleoside analogs selected from the group consisting of cyclohexene-2 deoxyriboside, cyclohexene-2-deoxyribose, de claim 2 -deoxyriboside, and benzene-2 deoxyriboside wherein said one or more non-florescent nucleoside analogs is interspaced between the fluorescent nucleoside analogs; and 
       (b) hybridizing a nucleic acid comprising a donor or acceptor dye to a nucleic acid sequence in the CFA library and (c)correlating any change in color exhibited by the hybridized molecules with energy transfer.  
     
     
       24. The method according to  claim 23  wherein the sugar moiety is ribose or deoxyribose. 
     
     
       25. The method according to  claim 23  wherein the fluorescent cyclic compound is an oligomer of varying length selected from the group consisting of oligothiophene, oligobenzothiophene, oligo(phenylene vinylene), and oligo(phenylene acetylene). 
     
     
       26. The method according to  claim 25  wherein the fluorescent cyclic compound has an oligomer length of from about 1 to about 16. 
     
     
       27. The method according to  claim 25  wherein the oligothiophene is a terthiophene or a sexithiophene. 
     
     
       28. The method according to  claim 25  wherein the oligobenzothiophene is a benzoterthiophene or a terbenzothiophene. 
     
     
       29. The method according to  claim 25  wherein the oligo(phenylene vinylene) is dimethylamino stilbene or styrylstilbene. 
     
     
       30. The method according to  claim 25  wherein the oligo(phenylene acetylene) is diphenylacetylene or phenyl(ethynyl)diphenylacetylene. 
     
     
       31. The method according to  claim 23  wherein the fluorescent cyclic compound is selected from the group consisting of p-terphenyl, perylene, azobenzene, phenazine, napthalene, phenanthroline, acridine, thioxanthrene, chrysene, rubrene, coronene, cyanine, perylene imide, and perylene amide. 
     
     
       32. The method of identifying a fluorophore involved in energy transfer which comprises: 
       (a) constructing a combinatorial fluorophore array library (CFA) which comprises multiple solid supports or multiple locations on a solid support, wherein each support location has attached thereto an oligomer comprising a nucleoside analog, said nucleoside analog comprising a fluorescent cyclic compound joined at the C-1 position of a sugar moiety in either an α or β configuration wherein said sugar moiety is a pentose or hexose, and one or more non-fluorescent nucleoside analogs wherein the one or more non-fluorescent nucleosides are positioned 5′ or 3′ to the fluorescent cyclic compound or interspaced between the fluorescent cyclic compound or between the fluorescent cyclic compound and the unlabelled nucleoside and wherein one or more non-fluorescent nucleoside analog is selected from the group selected from cyclohexene-2-deoxyriboside, cyclohexane-2-deoxyriboside, decalin-2-deoxyriboside, and benzene-2-deoxyriboside between the fluorescent nucleoside analogs or between the fluorescent nucleoside analog;  
       (b) hybridizing a nucleic acid comprising a donor or acceptor dye to a nucleic acid sequence in the CFA library; and  
       (c) correlating any change in color exhibited by the hybridized molecules with energy transfer.  
     
     
       33. A method for identifying a fluorophore sequence that changes its emission wavelength or intensity on binding an analyte, which method comprises: 
       (a) constructing a combinatorial fluorophore array library which comprises multiple solid supports or multiple locations on a solid support, wherein each support or location has attached thereto an oligomer comprising a nucleoside analog, said nucleoside analog being a fluorescent cyclic compound joined to a carbon at the C1 position of a sugar moiety in either an α or β configuration wherein said sugar moiety is a pentose or hexose;  
       (b) incorporating an analyte affinity molecule;  
       (c) allowing an analyte solution to contact the library; and  
       (d) selecting library members that change emission wavelength intensity or wavelength on binding of the analyte molecule.  
     
     
       34. The method according to  claim 33  wherein the sugar moiety is a ribose of deoxyribose. 
     
     
       35. The method according to  claim 33  wherein the fluorescent cyclic compound is an oligomer of varying length selected from the group consisting of oligothiophene, oligobenzothiophene, oligo(phenylene vinylene), and oligo(phenylene acetylene). 
     
     
       36. The method according to  claim 35  wherein the fluorescent cyclic compound has an oligomer length of from about 1 to about 16. 
     
     
       37. The method according to  claim 35  wherein the oligothiophene is a terthiophene or a sexithiophene. 
     
     
       38. The method according to  claim 35  wherein the oligobenzothiophene is a benzoterthiophene or a terbenzothiophene. 
     
     
       39. The method according to  claim 35  wherein the oligo(phenylene vinylene) is dimethylamino stilbene or styrylstilbene. 
     
     
       40. The method according to  claim 35  the oligo(phenylene acetylene) is diphenylacetylene or phenyl(ethynyl)diphenylacetylene. 
     
     
       41. The method according to  claim 33  wherein the fluorescent cyclic compound is selected from the group consisting of p-terphenyl, perylene, azobenzene, phenazine, napthalene, phenanthroline, acridine, thioxanthrene, chrysene, rubrene, coronene, cyanine, perylene imide, and perylene amide. 
     
     
       42. The method for identifying a fluorophore sequence that changes its emission wavelength or intensity on binding an analyte according to an one of claims  33 - 41  wherein the combinatorial fluorophore array library further comprises one or more unlabeled nucleosides wherein the one or more unlabeled nucleosides are positioned 5′ or 3′ to the fluorescent nucleoside analogs or interspaced between the fluorescent nucleoside analogs. 
     
     
       43. The method according to any one of claims  33 - 41  wherein the CFA library further comprises one or more non-fluorescent nucleoside analogs selected from the group consisting of cyclohexene-2-deoxyriboside, cyclohexane-2-deoxyriboside, decalin-2-deoxyriboside, and benzene-2-deoxyriboside wherein said one or more non-fluorescent nucleoside analogs are interspaced between the fluorescent nucleoside analogs. 
     
     
       44. The method according to  claim 42  wherein the CFA library further comprises one or more non-fluorescent nucleoside analogs selected from the group consisting of cyclohexene-2-deoxyriboside, cyclohexane-2-deoxyriboside, decalin-2-deoxyriboside, and benzene-2-deoxyriboside wherein said one or more non-fluorescent nucleoside analogs are interspaced between the fluorescent nucleoside analogs or between the fluorescent nucleoside analogs of the unlabeled nucleosides. 
     
     
       45. The method according to  claim 32  wherein the sugar moiety is ribose or deoxyribose. 
     
     
       46. The method according to  claim 32  wherein the fluorescent cyclic compound is an oligomer of varying length selected from the group consisting of oligothiophene, oligobenzothiophene, oligo(phenylene vinylene), and oligo(phenylene acetylene). 
     
     
       47. The method according to  claim 46  wherein the fluorescent cyclic compound has an oligomer length of from about 1 to about 16. 
     
     
       48. The method according to  claim 46  wherein the oligothiophene is a terthiophene or a sexithiophene. 
     
     
       49. The method according to  claim 46  wherein the oligobenzothiophene is a benzoterthiophene or a terbenzothiophene. 
     
     
       50. The method according to  claim 46  wherein the oligo(phenylene vinylene) is dimethylamino stilbene or styrylstilbene. 
     
     
       51. The method according to  claim 46  wherein the oligo(phenylene acetylene) is diphenylacetylene or phenyl(ethynyl)diphenylacetylene. 
     
     
       52. The method according to  claim 46  wherein the fluorescent cyclic compound is selected from the group consisting of p-terphenyl, perylene, azobenzene, phenazine, napthalene, phenanthroline, acridine, thioxanthrene, chrysene, rubrene, coronene, cyanine, perylene imide, and perylene amide.